This proposal is concerned with cellular interactions during development. The cell culture system described here, because of th opportunity of control of both the cellular and fluid environment of the neurons, may be useful in investigating both long-range, hormonal interactions as well as short-range signaling via cell contacts. Previous work from this laboratory documented some of the basic biochemical properties of sympathetic neurons when they are grown in the virtual absence of other cell types. These will be extended to include complete developmental curves for neuronal growth, catecholamine (CA) synthesis, uptake, storage, and release. More recently we have begun adding non-neuronal cells to the neurons. Unexpectedly, certain non-neuronal cell types, or medium conditioned (CM) by them, were found to cause the neuronal cultures to synthesize acetylcholine (ACh) as well as CA's. The neurons were also capable of forming cholinergic synapses under these conditions. The CM did not markedly affect neuronal survival, growth, or CA production and therefore appeared to be relatively specific in its action. Further studies on the CM-treated neurons are proposed to analyse in detail what other properties of the neurons may have changed. We plan to attempt purification of the active factor(s) causing cholinergic differentiation from serum-free CM. It will also be of interest to test a wider variety of cell types (glia-cholinergic and adrenergic targets, other neurons, etc.) for their ability to cause cholinergic differentiation in order to make generalizations if possible. Adrenergic parameters will also be monitoried in these studies so as to see any "trophic" effects of non-neuronal cells on CA metabolism. In addition to extending our studies in nerve-alone cultures of the effect of various concentrations of nerve growth factor on neuronal survival, growth, and biochemical differtiation, we propose investigation of various endocrine influences such as hydrocortisone and thyroxin on these parameters. These studies will be facilitated by the absence of non-neuronal cells which could alter the concentrations or effects of these hormones.